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Effects of photomirex and mirex on reproduction in the rat
TOXICOLOGY
AND
Effects
APPLIED
PHARMACOLOGY
of Photomirex
60,
549-556 (1981)
and Mirex on Reproduction
in the Rat
I. CHU, D. C. VILLENEUVE, V. E. SECOURS,V. E. VALLI,’ AND G. C. BECKING Environmental and Occupational Toxicology Division, Bureau of Chemical Hazards, Environmental Health Directorate, Ottawa, Ontario KIA OL2, Canada
Received
November
25, 1980; accepted
March
31. 1981
Effects of Photomirex and Mirex on Reproduction in the Rat. CHU, I., VILLENEUVE, V. E., VALLI,~. E., ANDBECKING, G. C. (1981). Toxicol. Appl. Pharmacol. 60, 549-556. Groups of weanling male and female rats were fed diets containing photomirex at levels of 0, 2.5, 5.0, 10, 20, or 40 ppm or mirex at 5.0, 10, 20, or 40 ppm for a period of 91 days prior to mating, 15 days during mating, and throughout gestation and lactation. Females fed 40 ppm photomirex or mirex diet showed a significant decrease in weight gain but not in food consumption. Both chemicals at levels of 5.0 ppm and up resulted in a decreased incidence of females showing sperm in vaginal smears. While the litter size was decreased in all treatment groups, the survival indices of pups were affected only by 40 ppm photomirex and 20 ppm mirex. On necropsy, enlarged livers were observed in adult females treated with 40 ppm of photomirex or mirex. Hepatic microsomal aminopyrine demethylase activities were increased in all treated groups of females as well as their offsprings. Minor biochemical changes in serum included elevated levels of cholesterol and total protein of female rats fed 40 ppm photomirex. Residue analysis showed that both chemicals accumulated in a dose-dependent manner in the fat and livers of the adult females, and in the livers of rat pups. None of the hematological parameters were affected by treatment. Histopathologic changes were observed in the livers and thyroids of mothers and pups from all treatment groups. Both photomirex and mirex caused cataract formation in the pups. D. C., SECOURS,
Photomirex (1,2,3,4,5,5,7,9,10,10-undecachlorohydropentacyclo(5 , 3 , 9.02*6.03~g.04*8) decane, or 8-monohydromirex, Fig. 1) residue has been identified in soil and wildlife samples (Hallett et al., 1976; Carlson et al., 1976). This compound is believed to be a major photodegradation product of mirex; the latter was used as an insecticide to control fire ants and as flame retardant in the manufacture of plastics. Like mirex, photomirex is resistant to biodegradation and remains in the adipose tissues of rats for an extended period of time (Chu et al., 1979). In the subchronic studies with rats carried out in our laboratories, photomirex was shown to cause mortality, hepatomegaly, fatty infiltration of the liver, induction of
’ Biopath Analysts Ltd., Guelph, Ontario, Canada.
microsomal enzymes, and thyroid and testicular lesions (Villeneuve er al., 1979a,b; Sundarum et al., 1980). Mirex has been shown to cause cataract in suckling rats and mice (Gaines and Kimbrough, 1970; Chernoff et al., 1976), and to increase the incidence of fetal abnormalities (Khera et al., 1976). In a teratogenicity study, photomirex administered to rabbits up to 10 mg/kg/day, did not produce any visceral and skeletal malformations in the pups (Villeneuve et al., 1979). In view of the high levels of both photomirex and mirex in the vertebrate food chain in south-central Ontario, the high human population density in this area and the effects of photomirex on testicular tissue, we undertook the present study to assess more fully the effects of these two chemicals on reproduction.
549
0041-008X/81/120549-08502.00/0 Copyright @ 1981 by Academic Press, Inc. All rights of reproduction in any form reswwd.
CHU ET AL.
550
PHOTOMIREX IS-MONOHYDROMIREX)
FIG. 1. Structures of mirex and photomirex.
METHODS Chemicals Mirex (purity > 98%) was obtained from Dr. L. S. Kaminsky, New York State Department of Health, Albany, New York, and was used without further purification. Photomirex was synthesized using a method described by Hallett et al. (1978). The purity of photomirex was 96% as is 3% of 2,8-dihydromirex and 1% unreacted mirex. All other chemicals were commercially available reagent grade materials. Animal Treatment Male and female weanling Sprague-Dawley rats (Biobreeding Laboratories, Montreal) were acclimated for 1 week before the start of the experiment. Groups of 15 males and 20 females were fed diets containing 0, 2.5, 5.0, 10, 20, or 40 ppm photomirex or 5.0, 10, 20, or 40 ppm mirex. The males and females were housed separately with 3 males and 4 females per common cage. The body weights and food consumption were measured on the females at weekly intervals. After 13 weeks of feeding 2 females were mated with 1 male for 15 days. Mating was carried out in order to obtain a minimum of 12 females from each group which showed sperm in vaginal smears. Male rats were discarded at this point since the effects of photomirex on males have been well documented (Villeneuve et al., 1979a.b). The groups of females were fed the same diets during and after mating and throughout gestation and lactation. After mating, the females were transferred to individual cages containing wood shavings for nesting. The pups were weighed at birth, and at 4 and 21 days after birth, and the numbers of surviving pups recorded. If there were sufficient number of pups, they were culled to eight per litter with an equal number of males and females. Twenty-one days after parturition adult females were anesthetized with ether and the blood was removed from the abdominal aorta. Hematological parameters examined included: hemoglobin concentration, packed cell volume, erythrocyte count, total and differential count of leukocytes, mean corpuscular volume, mean corpuscular hemoglobin concentration, and cytological evaluation of the bone marrow smears. The serum was an-
alyzed using a Technicon autoanalyzer (SMA 12160 micro) for sodium, potassium, inorganic phosphate, total bilirubin, alkaline phosphatase, glutamic oxalacetic transaminase (GOT), total protein, calcium, cholesterol, glucose, uric acid, and lactic dehydrogenase (LDH). In addition, y-glutamyltranspeptidase (Sigma Chemicals kit No. 415, 405 nm) and ornithine carbamoyl transferase activities (OCT, Vassef, 1978) were also measured. The brain, heart, liver, spleen, and kidneys were excised and weighed. A sample of the liver was homogenized in 0.05 mol Tris buffer/KC1 (pH 7.4) for the determination of microsomal aniline hydroxylase (AH, Fouts, 1963) and aminopyrine demethylase (APDM, Cochin and Axelrod, 1959) activities. All tissues were fixed in 10% phosphate-buffered formalin (pH 7.4) for histopathologic examination as described by Villeneuve er al. (1979a). The tissues examined included eye, brain, pituitary, liver, adrenal, thyroid, parathyroid, thymus, lungs, trachea, bronchi, thoracic aorta, esophagus, gastric cardia, fundus and pylorus, duodenum, jejunum, ileum, pancreas, colon, kidney, spleen, bone marrow, mesenteric and mediastinal lymph nodes, salivary glands, ovaries, uterus (for male pups testes, prostate, and seminal vesicles were also examined), heart, skeletal muscle, peripheral nerves, and skin. Samples of the liver and perirenal fat were taken for the residue analysis using a Hewlett-Packard gas chromatograph, Model 5830, equipped with a “Ni electron capture detector. The analytical procedure is described elsewhere (Hallett et al., 1978). Levels of photomirex and mirex were monitored in the diet throughout the study using a similar method. Pups that survived to 21 days of age were killed by an overdose of ether. When there were enough pups per litter, histopathologic examination, hepatic microsomal enzymes, and residue analysis, similar to those described for the adults, were carried out. Two pups were used for each determination. The priority of tests was given to histopathologic examination followed by microsomal enzymes and residue analysis. Determination of the total body burden for both chemicals is given below: whole pups were first frozen in liquid nitrogen, broken into small pieces with a Waring ice crusher, and the pieces were homogenized with an equal volume of water. The homogenates were analyzed in a manner similar to those described for the adult tissues. Statistical analysis of data was carried out using oneway analysis of variance (p 5 0.05) regression analysis, and the Duncan’s multiple-range test.
RESULTS Spontaneous death which occurred during the study included one male (2.5 ppm photomirex group) and five females: one, control; two, 40 ppm photomirex; one, 20 ppm mirex; and one, 40 ppm mirex. Clinical signs
index&
survival
index
index
index’
oi pups/lit-
stopped
pups thai was alive.
in parentheses.
in pregnancies.
are indicated
ol newborn
at p d 0.001.
’ The percenlage
’ Significanr
of animals
at p < 0.01.
C The number
resulting
dSignificant
of matings
o Percentage
when nmre than I2 females
k 5.5 (13)
48
Day 21
procedures
-c 0.9 (13)
98
o Mating
6.9 2 I.1 (13)
Day 4
per litw
97
97
97
10.2 k 4.4 (13)
;r 4.0 (12)
+ 7.8 (12)
2 I.1 (12)
IO/20
46
IO k6.1(9)
IN
RATS
IO
50
IO
0.98 (9)
smears.
+ 68 (9)
f
7.0 -f- I.5 (9)
98
100
100
6.2 ‘- 3.7 (9)’
90 (9/10)
IO/20
(ppm)
in vaginal
lk 1.7 (9)
7.2 + 1.6 (9)
9s
95
IO0
6.2 2 3.9 (9)’
90 (9/10)
5.0
Photomirex
PARAMETERS
from each group showed sperms
49
IO
6.8 + 0.8 (12)
95
95
97
85
13/14
loo (13/13)
14/14
2.5
loo (14/14)
Day I
+SD)
of the average pup weight
Mean body weigh! of pups (g. mean
survival
21-Day
survival
4-Day
Gestational
ter) -tSD
sperms in va-
of females used
Mean httcr size (number
Fertility
for mating
ginal smeaP/No
No. of females showing
0
REPRODUCTIVE
TABLE
1
O/8)
48
IO
1.6 (7)
+ 36 (7)
k 0
10.5 (I)
7.2 2 0.8 (4)
w
75
6.6 _+ ).I (8)
79” 36’
2.3 ? I.2 (6)
I00 (6/6)
6120
40
OR MIREX
92 89
6.9 + 5.2 (LW
loo
8120
20
FED PHOTOMIREX
5.2
I2
I.2 (IO) k 2.9 (IO)
t
7.2 f 0.6 (IO)
96
99 96
7.0 + 3.4 (I l)d
loo(ll/ll)
II/20
5.0
(WI
(ppm)
46
+ IO(S)
9.9 + 2.2 (5)
6.5 + 0.9 (6)
84
92 89
5.8 ? 3.8 (6)d
100
6120
IO
Mirex
45
+ 6.6 (6)
9.3 + 1.8 (7)
6.8 + 0.5 (7)
7w
96 96
7.1 + 4.2 (7)=’
loo (717)
7/20
20
-
-
-
-
-
-
o/20 -
40
2.1 * 9.0 k 14.9 +
Photomirex 2.5 5.0 10.0
5.1 * 1.2 (5) 13.5 + 3.4 (5) 32.1 k 11.4 (5) 120 (1)
Mirex 5.0 10.0 20.0 40.0
37 (5) 34 (5) 54 (5)
311 rt_ 68 (5) 417 + 130 (5) 862 + 220 (5) 730 (1)
965 k 281 (5) 2190 k 141 (3)
106 + 190 * 539 +
0 (5)
Fat 0.02 + 0.04 (5)
+ 2.9 (3) -
14.3 * 1.0 (3) 23 2 3.6 (3) 53 (1) -
66
8.5 f 1.8 (5) 14.9 -c 0.9 (5) 35 zk 4.6 (4)
PRESENT
TISSUES”
Liver
IN RAT
105
16.1
114
2 + -
7.5 (3)
8.5 (5)
f 23 (4) -
16.5 -t 2.6 (5) 42.0 k 11.1 (5) 72 * 15.4 (5)
0.07 k 0.04 (5)
Male pups6
Total body burden
RESIDUES
2
’ Mean k SD ppm based on wet tissues; numbers of the animals are indicated in parentheses. ’ Pups were nursed by the mothers and had no direct access to the diets. ’ No pups survived for 21 days.
35.1 + 14.3 (5) 134 k 64 (3)
20.0 4O.W
0.4 (5) 6.6 (5) 6.0 (5)
0.04 k 0.07 (5)
Liver
Females
0 (control)
Treatment level of chemical in diet (ppm)
PHOTOMIREX AND MIREX
TABLE
12 31 55
67
-
7.2 (5)
k 2.6 (5) -e 66 (5) + 5.8 (5) -
f
7.2 -+ 0.58 (5) 12.7 -+ 3.0 (5) 35 -+ 5.0 (5)
0.08 + 0.13 (5)
Total body burden
Liver
15.6 34 83
115
19.7 38 76
r 3.7 (5) -+ 8.9 (5) + 26 (5) -
+ 36 (5) -
-+ 2.3 (5) 1- 6.0 (5) +- 30 (5)
0.04 -I- 0.02 (5)
Female pup?
F
7
PHOTOMIREX
AND MIREX
of toxicity such as hypoactivity, irritability and muscle tremor were manifested in the highest treatment groups of photomirex and mirex. The food consumption of the preg-
IN THE RAT
nant females was not affected by treatment. However, the weight gains of the female rats receiving 40 ppm photomirex or mirex diet were significantly depressed. Weight
Initial
weight (g)
Control: 92 -t 8 g (13) 40 ppm photomirex: 94 + 6 (3) 40 ppm mirex: 98 ( 1)
553
gain measured before necropsy (8)
Control: 195 k 26 (13) 40 ppm photomirex: 143 -+ 8 (3) 40 ppm mirex: 108 ( 1)
a Values represent mean k SD from the numbers of animals shown in parentheses.
The effects of photomirex and mirex on the reproductive performances of female adults and neonatal developments are presented in Table 1. The chemicals caused a dose-related decrease in the number of females which showed sperm in vaginal smears. However, the fertility index was not affected. A number of adverse effects was manifested in the pups. The litter size was decreased in all treatment groups and the survival indices declined in the 40-ppm-photomirex and 20-ppm-mirex group. Analysis of the effect of treatment on pup weights was complicated by variations in litter size. The litter sizes of the treated groups were significantly smaller than that of the control. The reduction in litter size would lessen the competition of the treated pups for nourishment from lactating mothers. Consequently, the growth retardation exerted by the compounds may have been compensated for by an increase in milk availability. The neonate body weights may have been biased by the treatment-related effect on litter size. Gross pathologic examination showed that the female adults on the highest levels of photomirex and mirex diet had enlarged livers. The liver weights expressed as a percentage of body weights were increased (control, 4.4 t- 0.49 (13); 40 ppm photomirex, 7.1 & 0.55 (3); 40 ppm mirex, 8.4 (l), mean 2 SD from the numbers shown in parentheses). The weights of the other organs were not affected by treatment.
Tissue residue data are presented in Table 2. Dose-dependent accumulation of the chemicals was observed in the liver and fat of the females and in the liver of the pups. Both mirex and photomirex appeared to accumulate to the same extent in the maternal tissues. The levels of photomirex in the pup liver were three- to fourfold higher than those of the corresponding maternal tissues, and those of mirex were two- to threefold higher. Alterations in serum biochemical profiles were considered minor in nature, and included increased levels of cholesterol (control,71 + 19mg/lOOml;treated, 167 +- 114 mg/IOO ml) and total protein (control, 6.0
l T
CONTROL
2.5u) la 20 40 PHOTOMIREX
5.0 IO 20 (pQml MIREX
FIG. 2. Effects of photomirex and mirex on the hepatic microsomal aminopyrine demethylase activities of adult female rats. *Significantly different from control (p c 0.05).
CHU ET AL.
554
TABLE
3
PREVALENCE OF LESSONS IN RATS FED PHOTOMIREX Treatment levels of chemical in the diet (ppm) 0 (control) Photomirex 2.5 5.0 10.0 20 40 Mirex 5.0 10.0 20 40
Female adults
Female pups
Male pups
Liver
Thyroid
Liver
Thyroid
Eye
2113
2113
l/IO
O/l0
O/IO
5112 6/Q 919 718 ‘/’
2/12 519 219 5/g ‘/’
*/g 4/5
l/8
O/8
414 -
l/5 3/o ‘14 -
015 ‘16 214 -
3/g 313 414 -
‘18 f/3 3/4 -
O/3 214 -
6110
616 2/b ‘I’
OR MIREX”
616
018
Liver
Thyroid
Eyeb
2114
O/l4
O/l4
10/14 7/9 w 717 -
l/14 4/9 518 4/7 -
o/14 O/9 3/g 417 -
2/10 3/5
4flO
4110
2/5
z/5
b/7
417 -
4/7 -
-
’ Values denote: animals showing lesions/animals examined. b The presence of unilateral and bilateral cataracts was given an equal score.
& 0.33 g/100 ml; treated, 7.6 t- 2.2 g/100 ml) of female rats fed 40 ppm photomirex diet. A slight, but not statistically significant increase was observed in serum SDH of the 40-ppm-photomirex group (control, 8.7 + 7.3 mIU/ml; treated, 11.8 +- 11.1 mIU/ ml). Hepatic microsomal APDM activities were significantly increased in all groups fed photomirex and mirex (Fig. 2). Aniline hydroxylase activity was not significantly afl
w 2 40 m F v i a p
30
20
s P c ‘0
0 CONrROL
50 IO 20 tom) PHOTOMlREX
MIREX
.
FIG. 3. Effects of photomirex and mirex on the hepatic microsomal aminopyrine demethylase activities of Zlday-old neonates. *Significantly different from control @ < 0.05).
fected. APDM activities of all treated pups were increased (Fig. 3). Similar to their mothers, the hepatic AH activity of pups was not affected by treatment. No hematological values determined in females in any treatment groups were significantly different from controls. Histopathologic examination revealed that photomirex and mirex elicited lesions in the liver and thyroid of the female adults and pups, and in the eyes of the pups (Table 3). In female rats fed lower doses of the compounds, the morphological changes of the livers consisted of a mild lobular pattern, reduced cytoplasmic density of the hepatocytes, and increased perivenous cytoplasmic homogeneity and eosinophilia. The lesions became more severe as the dosage increased, and extended from the perivenous area to the midzone. Fatty infiltration, cytoplasmic vacuolations of hepatocytes, anisokaryosis, hyperchromicity, and reduction in basophilia were observed. Hepatic lesions in the pups were similar but were generally less severe than those observed in the adults. Lesions in the thyroid of adult females
PHOTOMIREX
AND
consisted of a reduction in colloid density and thickening of the follicular epithelia. Animals fed higher doses of the compounds exhibited angular collapse of the follicles with complete loss of the colloid. Lesions in the thyroids of pups were qualitatively similar to those in the adults but were less severe. A number of cataracts was observed in the pups of the treated groups starting from the third week of nursing. The cataract was central in type, and consisted of granular changes in the lens substance with vacuolation and focal mineralization. In the peripheral areas of the lens, there were increases in the nuclearity of the lens substance and anisokaryosis of the lenticular nuclei. Comparison of the severity of histopathologic lesions revealed that at 5.0- and loppm levels, mirex was more potent than photomirex in causing thyroid lesions and at higher dose levels, both compounds were equally potent. In contrast, photomirex was more potent than mirex in eliciting hepatic damage. Mirex was slightly more toxic than photomirex in producing cataracts in the pups (Table 4). DISCUSSION Results of the present study demonstrated that photomirex and mirex caused reproductive impairment in rats. Both chemicals at levels of 5.0 ppm and up resulted in a decreased incidence of animals showing sperm in vaginal smears. Photomirex at 40 ppm caused decreases in litter size and gestational, 4- and 21-day survival indices of pups. Khera et al. (1976) reported that mirex fed to female rats at 12.5 mg/kg on Days 6-15 of gestation caused pregnancy failure, decreased fetal survival rate and fetal visceral abnormalities. The cataractogenie effect of photomirex and mirex observed in this study is consistent with those of earlier results (Chernoff et al. 1976). Despite the fact that contaminants accumulated to a much higher level in the livers of rat pups than in their mothers, histopath-
MIREX
IN THE
RAT
CHU ET AL.
556
ologic changes were milder in pups than in the corresponding maternal tissues. Thus, the difference in the severity of hepatic lesions in the pups and mothers could not be related to the residue levels. Maternal toxicities observed here were similar to those of a 28- and 90-day study (Sundarum et al., 1980). In the 90-day study, hepatomegaly was elicited by 25 ppm photomirex and up as compared to 40 ppm in the present study. Results of serum biochemistry, hematology, and histopathologic examinations were also in general agreement with the previous study. Although AH activities were not significantly affected, there was a trend toward higher values when the dietary level of photomirex was increased. In comparison, mirex at the levels studied had no effect on AH activity. A decreased incidence of female rats showing sperm in vaginal smears could be attributed to a number of factors such as testicular damage in male rats as previously reported (Villeneuve et al., 1979a,b) and the health state of both male and female rats. Kepone, a structural analog of photomirex, is known to reduce luteinizing hormone activity in the pituitary of constant-estrous female mice, and may cause ovulation failure (Huber, 1965; Ware and Good, 1967). It was likely photomirex acted through a similar mechanism to reduce litter size in the treated groups. ACKNOWLEDGMENTS The authors thank Dr. I. A. Marino, A. P. Yagminas, N. Beament, B. Reed, A. Watt, M. Beaudette, and H. James for technical assistance, and D. Biggs for statistical analysis.
REFERENCES CARLSON, D. A., KANYHA, D. D., WHEELER, W. B., MARSHALL, G. P., AND ZAYLSKIE, R. G. (1976). Mirex in the environment: Its degradation to kepone and related compounds. Science 194, 939-941. CHERNOFF, N., Scorn, T. M., AND LINDER, R. E. (1976). Cataractogenic properties of mirex in rats and mice with notes on kepone. Toxicol. A@. Pharmacol. 37, 229A. CHU, I., VILLENEUVE, D. C., SECOURS, V., BECKING,
G. C., VIAU, A., AND BENOIT, F. (1979). The absorption, distribution and excretion of photomirex in the rat. Drug Merab. Dispos. 7, 24-27. COCHIN, J., AND AXELROD, J. (1959). Biochemical and pharmacological changes in the rat following chronic administration of morphine, nalorphine and normorphine. J. Pharmacol. Exp. Ther. 125, 105-l 10. Fours, J. R. (1963). Factors influencing the metabolism of drugs in liver microsomes. Ann. N.Y. Acad. Sci. 104, 875-880. GAINES, T. B., AND KIMBROUGH, R. (1970). Oral toxicity of mirex in adult and suckling rats. Arch. Environ. Health 21, l-14. HALLETI-, D. J., NORSTROM, R. J., ONUSKA, F. I., COMBA, M. E., AND SAMPSON, R. (1976). Mass spectral confirmation and analysis by the Hall detector of mirex and photomirex in herring gulls from Lake Ontario. .I. Agr. Food Chem. 24, 1189-l 193. HALLETT, D. J., KHERA, K. S., STOLTZ, D. R., CHU, I., VILLENEUVE, D. C., AND TRIVETT, G. (1978). Photomirex: Synthesis and assessment of acute toxicity, tissue distribution, and mutagenicity. J. Agr. Food Chem. 26, 388-391. HUBER, J. J. (1965). Some physiological effects of the insecticide kepone in the laboratory mouse. Toxicol. Appt. Pharmacol. 7, 516-524. KHERA, K. S., VILLENEUVE, D. C., TERRY, G., PANOPIO, L., NASH, L., AND TRIVETT, G. (1976). Mirex: A teratogenicity, dominant lethal and tissue distribution study in rats. Food Cosmet. Toxicol. 14, 2529. SUNDARUM, A., VILLENEUVE, D. C., CHU, I., SECOURS,V. BECKING, G. C.. AND VALLI, V. E. (1980). Subchronic toxicity of photomirex in the female rat: Results of 28- and 90-day feeding studies. Drug Chem. Toxicol. 3, 105- 134. VASSEF, A. A. (1978). Direct micromethcd for colorimetry of some ornithine cambamoyl transferase activity with use of a linear standard curve. Clin. Chem. 24, 101-107. VILLENEUVE, D. C., RITTER, L., FELSKY, G., NORSTROM, R. J., MARINO, 1. A., VALLI, V. E., CHU, I., AND BECKING, G. C. (1979a). Short-term toxicity of photomirex in the rat. Toxicol. Appi. Pharmacol. 47, 105-I 14. VILLENEUVE, D. C., VALLI, V. E., CHU, I., SECOURS, V., RITTER, L., AND BECKING, G. C. (1979b). Ninety-day toxicity of photomirex in the male rat. Toxicology 12, 235-250. VILLENEUVE, D. C., KHERA, K. S., TRIVETT, G.. FELSKY, G., NORSTROM, R. J., AND CHU, 1. (1980). Photomirex: A tetratogenicity and tissue distribution study in the rabbit. J. Environ. Health Sci. 2, 171180. WARE, G. W., AND Goon, E. E. (1967). Effects of insecticides on reproduction in the laboratory mouse. II. Mirex, Tel&in, and DDT. Toxicol. Appl. Pharmacol. 10, 54-61.
AND
Effects
APPLIED
PHARMACOLOGY
of Photomirex
60,
549-556 (1981)
and Mirex on Reproduction
in the Rat
I. CHU, D. C. VILLENEUVE, V. E. SECOURS,V. E. VALLI,’ AND G. C. BECKING Environmental and Occupational Toxicology Division, Bureau of Chemical Hazards, Environmental Health Directorate, Ottawa, Ontario KIA OL2, Canada
Received
November
25, 1980; accepted
March
31. 1981
Effects of Photomirex and Mirex on Reproduction in the Rat. CHU, I., VILLENEUVE, V. E., VALLI,~. E., ANDBECKING, G. C. (1981). Toxicol. Appl. Pharmacol. 60, 549-556. Groups of weanling male and female rats were fed diets containing photomirex at levels of 0, 2.5, 5.0, 10, 20, or 40 ppm or mirex at 5.0, 10, 20, or 40 ppm for a period of 91 days prior to mating, 15 days during mating, and throughout gestation and lactation. Females fed 40 ppm photomirex or mirex diet showed a significant decrease in weight gain but not in food consumption. Both chemicals at levels of 5.0 ppm and up resulted in a decreased incidence of females showing sperm in vaginal smears. While the litter size was decreased in all treatment groups, the survival indices of pups were affected only by 40 ppm photomirex and 20 ppm mirex. On necropsy, enlarged livers were observed in adult females treated with 40 ppm of photomirex or mirex. Hepatic microsomal aminopyrine demethylase activities were increased in all treated groups of females as well as their offsprings. Minor biochemical changes in serum included elevated levels of cholesterol and total protein of female rats fed 40 ppm photomirex. Residue analysis showed that both chemicals accumulated in a dose-dependent manner in the fat and livers of the adult females, and in the livers of rat pups. None of the hematological parameters were affected by treatment. Histopathologic changes were observed in the livers and thyroids of mothers and pups from all treatment groups. Both photomirex and mirex caused cataract formation in the pups. D. C., SECOURS,
Photomirex (1,2,3,4,5,5,7,9,10,10-undecachlorohydropentacyclo(5 , 3 , 9.02*6.03~g.04*8) decane, or 8-monohydromirex, Fig. 1) residue has been identified in soil and wildlife samples (Hallett et al., 1976; Carlson et al., 1976). This compound is believed to be a major photodegradation product of mirex; the latter was used as an insecticide to control fire ants and as flame retardant in the manufacture of plastics. Like mirex, photomirex is resistant to biodegradation and remains in the adipose tissues of rats for an extended period of time (Chu et al., 1979). In the subchronic studies with rats carried out in our laboratories, photomirex was shown to cause mortality, hepatomegaly, fatty infiltration of the liver, induction of
’ Biopath Analysts Ltd., Guelph, Ontario, Canada.
microsomal enzymes, and thyroid and testicular lesions (Villeneuve er al., 1979a,b; Sundarum et al., 1980). Mirex has been shown to cause cataract in suckling rats and mice (Gaines and Kimbrough, 1970; Chernoff et al., 1976), and to increase the incidence of fetal abnormalities (Khera et al., 1976). In a teratogenicity study, photomirex administered to rabbits up to 10 mg/kg/day, did not produce any visceral and skeletal malformations in the pups (Villeneuve et al., 1979). In view of the high levels of both photomirex and mirex in the vertebrate food chain in south-central Ontario, the high human population density in this area and the effects of photomirex on testicular tissue, we undertook the present study to assess more fully the effects of these two chemicals on reproduction.
549
0041-008X/81/120549-08502.00/0 Copyright @ 1981 by Academic Press, Inc. All rights of reproduction in any form reswwd.
CHU ET AL.
550
PHOTOMIREX IS-MONOHYDROMIREX)
FIG. 1. Structures of mirex and photomirex.
METHODS Chemicals Mirex (purity > 98%) was obtained from Dr. L. S. Kaminsky, New York State Department of Health, Albany, New York, and was used without further purification. Photomirex was synthesized using a method described by Hallett et al. (1978). The purity of photomirex was 96% as is 3% of 2,8-dihydromirex and 1% unreacted mirex. All other chemicals were commercially available reagent grade materials. Animal Treatment Male and female weanling Sprague-Dawley rats (Biobreeding Laboratories, Montreal) were acclimated for 1 week before the start of the experiment. Groups of 15 males and 20 females were fed diets containing 0, 2.5, 5.0, 10, 20, or 40 ppm photomirex or 5.0, 10, 20, or 40 ppm mirex. The males and females were housed separately with 3 males and 4 females per common cage. The body weights and food consumption were measured on the females at weekly intervals. After 13 weeks of feeding 2 females were mated with 1 male for 15 days. Mating was carried out in order to obtain a minimum of 12 females from each group which showed sperm in vaginal smears. Male rats were discarded at this point since the effects of photomirex on males have been well documented (Villeneuve et al., 1979a.b). The groups of females were fed the same diets during and after mating and throughout gestation and lactation. After mating, the females were transferred to individual cages containing wood shavings for nesting. The pups were weighed at birth, and at 4 and 21 days after birth, and the numbers of surviving pups recorded. If there were sufficient number of pups, they were culled to eight per litter with an equal number of males and females. Twenty-one days after parturition adult females were anesthetized with ether and the blood was removed from the abdominal aorta. Hematological parameters examined included: hemoglobin concentration, packed cell volume, erythrocyte count, total and differential count of leukocytes, mean corpuscular volume, mean corpuscular hemoglobin concentration, and cytological evaluation of the bone marrow smears. The serum was an-
alyzed using a Technicon autoanalyzer (SMA 12160 micro) for sodium, potassium, inorganic phosphate, total bilirubin, alkaline phosphatase, glutamic oxalacetic transaminase (GOT), total protein, calcium, cholesterol, glucose, uric acid, and lactic dehydrogenase (LDH). In addition, y-glutamyltranspeptidase (Sigma Chemicals kit No. 415, 405 nm) and ornithine carbamoyl transferase activities (OCT, Vassef, 1978) were also measured. The brain, heart, liver, spleen, and kidneys were excised and weighed. A sample of the liver was homogenized in 0.05 mol Tris buffer/KC1 (pH 7.4) for the determination of microsomal aniline hydroxylase (AH, Fouts, 1963) and aminopyrine demethylase (APDM, Cochin and Axelrod, 1959) activities. All tissues were fixed in 10% phosphate-buffered formalin (pH 7.4) for histopathologic examination as described by Villeneuve er al. (1979a). The tissues examined included eye, brain, pituitary, liver, adrenal, thyroid, parathyroid, thymus, lungs, trachea, bronchi, thoracic aorta, esophagus, gastric cardia, fundus and pylorus, duodenum, jejunum, ileum, pancreas, colon, kidney, spleen, bone marrow, mesenteric and mediastinal lymph nodes, salivary glands, ovaries, uterus (for male pups testes, prostate, and seminal vesicles were also examined), heart, skeletal muscle, peripheral nerves, and skin. Samples of the liver and perirenal fat were taken for the residue analysis using a Hewlett-Packard gas chromatograph, Model 5830, equipped with a “Ni electron capture detector. The analytical procedure is described elsewhere (Hallett et al., 1978). Levels of photomirex and mirex were monitored in the diet throughout the study using a similar method. Pups that survived to 21 days of age were killed by an overdose of ether. When there were enough pups per litter, histopathologic examination, hepatic microsomal enzymes, and residue analysis, similar to those described for the adults, were carried out. Two pups were used for each determination. The priority of tests was given to histopathologic examination followed by microsomal enzymes and residue analysis. Determination of the total body burden for both chemicals is given below: whole pups were first frozen in liquid nitrogen, broken into small pieces with a Waring ice crusher, and the pieces were homogenized with an equal volume of water. The homogenates were analyzed in a manner similar to those described for the adult tissues. Statistical analysis of data was carried out using oneway analysis of variance (p 5 0.05) regression analysis, and the Duncan’s multiple-range test.
RESULTS Spontaneous death which occurred during the study included one male (2.5 ppm photomirex group) and five females: one, control; two, 40 ppm photomirex; one, 20 ppm mirex; and one, 40 ppm mirex. Clinical signs
index&
survival
index
index
index’
oi pups/lit-
stopped
pups thai was alive.
in parentheses.
in pregnancies.
are indicated
ol newborn
at p d 0.001.
’ The percenlage
’ Significanr
of animals
at p < 0.01.
C The number
resulting
dSignificant
of matings
o Percentage
when nmre than I2 females
k 5.5 (13)
48
Day 21
procedures
-c 0.9 (13)
98
o Mating
6.9 2 I.1 (13)
Day 4
per litw
97
97
97
10.2 k 4.4 (13)
;r 4.0 (12)
+ 7.8 (12)
2 I.1 (12)
IO/20
46
IO k6.1(9)
IN
RATS
IO
50
IO
0.98 (9)
smears.
+ 68 (9)
f
7.0 -f- I.5 (9)
98
100
100
6.2 ‘- 3.7 (9)’
90 (9/10)
IO/20
(ppm)
in vaginal
lk 1.7 (9)
7.2 + 1.6 (9)
9s
95
IO0
6.2 2 3.9 (9)’
90 (9/10)
5.0
Photomirex
PARAMETERS
from each group showed sperms
49
IO
6.8 + 0.8 (12)
95
95
97
85
13/14
loo (13/13)
14/14
2.5
loo (14/14)
Day I
+SD)
of the average pup weight
Mean body weigh! of pups (g. mean
survival
21-Day
survival
4-Day
Gestational
ter) -tSD
sperms in va-
of females used
Mean httcr size (number
Fertility
for mating
ginal smeaP/No
No. of females showing
0
REPRODUCTIVE
TABLE
1
O/8)
48
IO
1.6 (7)
+ 36 (7)
k 0
10.5 (I)
7.2 2 0.8 (4)
w
75
6.6 _+ ).I (8)
79” 36’
2.3 ? I.2 (6)
I00 (6/6)
6120
40
OR MIREX
92 89
6.9 + 5.2 (LW
loo
8120
20
FED PHOTOMIREX
5.2
I2
I.2 (IO) k 2.9 (IO)
t
7.2 f 0.6 (IO)
96
99 96
7.0 + 3.4 (I l)d
loo(ll/ll)
II/20
5.0
(WI
(ppm)
46
+ IO(S)
9.9 + 2.2 (5)
6.5 + 0.9 (6)
84
92 89
5.8 ? 3.8 (6)d
100
6120
IO
Mirex
45
+ 6.6 (6)
9.3 + 1.8 (7)
6.8 + 0.5 (7)
7w
96 96
7.1 + 4.2 (7)=’
loo (717)
7/20
20
-
-
-
-
-
-
o/20 -
40
2.1 * 9.0 k 14.9 +
Photomirex 2.5 5.0 10.0
5.1 * 1.2 (5) 13.5 + 3.4 (5) 32.1 k 11.4 (5) 120 (1)
Mirex 5.0 10.0 20.0 40.0
37 (5) 34 (5) 54 (5)
311 rt_ 68 (5) 417 + 130 (5) 862 + 220 (5) 730 (1)
965 k 281 (5) 2190 k 141 (3)
106 + 190 * 539 +
0 (5)
Fat 0.02 + 0.04 (5)
+ 2.9 (3) -
14.3 * 1.0 (3) 23 2 3.6 (3) 53 (1) -
66
8.5 f 1.8 (5) 14.9 -c 0.9 (5) 35 zk 4.6 (4)
PRESENT
TISSUES”
Liver
IN RAT
105
16.1
114
2 + -
7.5 (3)
8.5 (5)
f 23 (4) -
16.5 -t 2.6 (5) 42.0 k 11.1 (5) 72 * 15.4 (5)
0.07 k 0.04 (5)
Male pups6
Total body burden
RESIDUES
2
’ Mean k SD ppm based on wet tissues; numbers of the animals are indicated in parentheses. ’ Pups were nursed by the mothers and had no direct access to the diets. ’ No pups survived for 21 days.
35.1 + 14.3 (5) 134 k 64 (3)
20.0 4O.W
0.4 (5) 6.6 (5) 6.0 (5)
0.04 k 0.07 (5)
Liver
Females
0 (control)
Treatment level of chemical in diet (ppm)
PHOTOMIREX AND MIREX
TABLE
12 31 55
67
-
7.2 (5)
k 2.6 (5) -e 66 (5) + 5.8 (5) -
f
7.2 -+ 0.58 (5) 12.7 -+ 3.0 (5) 35 -+ 5.0 (5)
0.08 + 0.13 (5)
Total body burden
Liver
15.6 34 83
115
19.7 38 76
r 3.7 (5) -+ 8.9 (5) + 26 (5) -
+ 36 (5) -
-+ 2.3 (5) 1- 6.0 (5) +- 30 (5)
0.04 -I- 0.02 (5)
Female pup?
F
7
PHOTOMIREX
AND MIREX
of toxicity such as hypoactivity, irritability and muscle tremor were manifested in the highest treatment groups of photomirex and mirex. The food consumption of the preg-
IN THE RAT
nant females was not affected by treatment. However, the weight gains of the female rats receiving 40 ppm photomirex or mirex diet were significantly depressed. Weight
Initial
weight (g)
Control: 92 -t 8 g (13) 40 ppm photomirex: 94 + 6 (3) 40 ppm mirex: 98 ( 1)
553
gain measured before necropsy (8)
Control: 195 k 26 (13) 40 ppm photomirex: 143 -+ 8 (3) 40 ppm mirex: 108 ( 1)
a Values represent mean k SD from the numbers of animals shown in parentheses.
The effects of photomirex and mirex on the reproductive performances of female adults and neonatal developments are presented in Table 1. The chemicals caused a dose-related decrease in the number of females which showed sperm in vaginal smears. However, the fertility index was not affected. A number of adverse effects was manifested in the pups. The litter size was decreased in all treatment groups and the survival indices declined in the 40-ppm-photomirex and 20-ppm-mirex group. Analysis of the effect of treatment on pup weights was complicated by variations in litter size. The litter sizes of the treated groups were significantly smaller than that of the control. The reduction in litter size would lessen the competition of the treated pups for nourishment from lactating mothers. Consequently, the growth retardation exerted by the compounds may have been compensated for by an increase in milk availability. The neonate body weights may have been biased by the treatment-related effect on litter size. Gross pathologic examination showed that the female adults on the highest levels of photomirex and mirex diet had enlarged livers. The liver weights expressed as a percentage of body weights were increased (control, 4.4 t- 0.49 (13); 40 ppm photomirex, 7.1 & 0.55 (3); 40 ppm mirex, 8.4 (l), mean 2 SD from the numbers shown in parentheses). The weights of the other organs were not affected by treatment.
Tissue residue data are presented in Table 2. Dose-dependent accumulation of the chemicals was observed in the liver and fat of the females and in the liver of the pups. Both mirex and photomirex appeared to accumulate to the same extent in the maternal tissues. The levels of photomirex in the pup liver were three- to fourfold higher than those of the corresponding maternal tissues, and those of mirex were two- to threefold higher. Alterations in serum biochemical profiles were considered minor in nature, and included increased levels of cholesterol (control,71 + 19mg/lOOml;treated, 167 +- 114 mg/IOO ml) and total protein (control, 6.0
l T
CONTROL
2.5u) la 20 40 PHOTOMIREX
5.0 IO 20 (pQml MIREX
FIG. 2. Effects of photomirex and mirex on the hepatic microsomal aminopyrine demethylase activities of adult female rats. *Significantly different from control (p c 0.05).
CHU ET AL.
554
TABLE
3
PREVALENCE OF LESSONS IN RATS FED PHOTOMIREX Treatment levels of chemical in the diet (ppm) 0 (control) Photomirex 2.5 5.0 10.0 20 40 Mirex 5.0 10.0 20 40
Female adults
Female pups
Male pups
Liver
Thyroid
Liver
Thyroid
Eye
2113
2113
l/IO
O/l0
O/IO
5112 6/Q 919 718 ‘/’
2/12 519 219 5/g ‘/’
*/g 4/5
l/8
O/8
414 -
l/5 3/o ‘14 -
015 ‘16 214 -
3/g 313 414 -
‘18 f/3 3/4 -
O/3 214 -
6110
616 2/b ‘I’
OR MIREX”
616
018
Liver
Thyroid
Eyeb
2114
O/l4
O/l4
10/14 7/9 w 717 -
l/14 4/9 518 4/7 -
o/14 O/9 3/g 417 -
2/10 3/5
4flO
4110
2/5
z/5
b/7
417 -
4/7 -
-
’ Values denote: animals showing lesions/animals examined. b The presence of unilateral and bilateral cataracts was given an equal score.
& 0.33 g/100 ml; treated, 7.6 t- 2.2 g/100 ml) of female rats fed 40 ppm photomirex diet. A slight, but not statistically significant increase was observed in serum SDH of the 40-ppm-photomirex group (control, 8.7 + 7.3 mIU/ml; treated, 11.8 +- 11.1 mIU/ ml). Hepatic microsomal APDM activities were significantly increased in all groups fed photomirex and mirex (Fig. 2). Aniline hydroxylase activity was not significantly afl
w 2 40 m F v i a p
30
20
s P c ‘0
0 CONrROL
50 IO 20 tom) PHOTOMlREX
MIREX
.
FIG. 3. Effects of photomirex and mirex on the hepatic microsomal aminopyrine demethylase activities of Zlday-old neonates. *Significantly different from control @ < 0.05).
fected. APDM activities of all treated pups were increased (Fig. 3). Similar to their mothers, the hepatic AH activity of pups was not affected by treatment. No hematological values determined in females in any treatment groups were significantly different from controls. Histopathologic examination revealed that photomirex and mirex elicited lesions in the liver and thyroid of the female adults and pups, and in the eyes of the pups (Table 3). In female rats fed lower doses of the compounds, the morphological changes of the livers consisted of a mild lobular pattern, reduced cytoplasmic density of the hepatocytes, and increased perivenous cytoplasmic homogeneity and eosinophilia. The lesions became more severe as the dosage increased, and extended from the perivenous area to the midzone. Fatty infiltration, cytoplasmic vacuolations of hepatocytes, anisokaryosis, hyperchromicity, and reduction in basophilia were observed. Hepatic lesions in the pups were similar but were generally less severe than those observed in the adults. Lesions in the thyroid of adult females
PHOTOMIREX
AND
consisted of a reduction in colloid density and thickening of the follicular epithelia. Animals fed higher doses of the compounds exhibited angular collapse of the follicles with complete loss of the colloid. Lesions in the thyroids of pups were qualitatively similar to those in the adults but were less severe. A number of cataracts was observed in the pups of the treated groups starting from the third week of nursing. The cataract was central in type, and consisted of granular changes in the lens substance with vacuolation and focal mineralization. In the peripheral areas of the lens, there were increases in the nuclearity of the lens substance and anisokaryosis of the lenticular nuclei. Comparison of the severity of histopathologic lesions revealed that at 5.0- and loppm levels, mirex was more potent than photomirex in causing thyroid lesions and at higher dose levels, both compounds were equally potent. In contrast, photomirex was more potent than mirex in eliciting hepatic damage. Mirex was slightly more toxic than photomirex in producing cataracts in the pups (Table 4). DISCUSSION Results of the present study demonstrated that photomirex and mirex caused reproductive impairment in rats. Both chemicals at levels of 5.0 ppm and up resulted in a decreased incidence of animals showing sperm in vaginal smears. Photomirex at 40 ppm caused decreases in litter size and gestational, 4- and 21-day survival indices of pups. Khera et al. (1976) reported that mirex fed to female rats at 12.5 mg/kg on Days 6-15 of gestation caused pregnancy failure, decreased fetal survival rate and fetal visceral abnormalities. The cataractogenie effect of photomirex and mirex observed in this study is consistent with those of earlier results (Chernoff et al. 1976). Despite the fact that contaminants accumulated to a much higher level in the livers of rat pups than in their mothers, histopath-
MIREX
IN THE
RAT
CHU ET AL.
556
ologic changes were milder in pups than in the corresponding maternal tissues. Thus, the difference in the severity of hepatic lesions in the pups and mothers could not be related to the residue levels. Maternal toxicities observed here were similar to those of a 28- and 90-day study (Sundarum et al., 1980). In the 90-day study, hepatomegaly was elicited by 25 ppm photomirex and up as compared to 40 ppm in the present study. Results of serum biochemistry, hematology, and histopathologic examinations were also in general agreement with the previous study. Although AH activities were not significantly affected, there was a trend toward higher values when the dietary level of photomirex was increased. In comparison, mirex at the levels studied had no effect on AH activity. A decreased incidence of female rats showing sperm in vaginal smears could be attributed to a number of factors such as testicular damage in male rats as previously reported (Villeneuve et al., 1979a,b) and the health state of both male and female rats. Kepone, a structural analog of photomirex, is known to reduce luteinizing hormone activity in the pituitary of constant-estrous female mice, and may cause ovulation failure (Huber, 1965; Ware and Good, 1967). It was likely photomirex acted through a similar mechanism to reduce litter size in the treated groups. ACKNOWLEDGMENTS The authors thank Dr. I. A. Marino, A. P. Yagminas, N. Beament, B. Reed, A. Watt, M. Beaudette, and H. James for technical assistance, and D. Biggs for statistical analysis.
REFERENCES CARLSON, D. A., KANYHA, D. D., WHEELER, W. B., MARSHALL, G. P., AND ZAYLSKIE, R. G. (1976). Mirex in the environment: Its degradation to kepone and related compounds. Science 194, 939-941. CHERNOFF, N., Scorn, T. M., AND LINDER, R. E. (1976). Cataractogenic properties of mirex in rats and mice with notes on kepone. Toxicol. A@. Pharmacol. 37, 229A. CHU, I., VILLENEUVE, D. C., SECOURS, V., BECKING,
G. C., VIAU, A., AND BENOIT, F. (1979). The absorption, distribution and excretion of photomirex in the rat. Drug Merab. Dispos. 7, 24-27. COCHIN, J., AND AXELROD, J. (1959). Biochemical and pharmacological changes in the rat following chronic administration of morphine, nalorphine and normorphine. J. Pharmacol. Exp. Ther. 125, 105-l 10. Fours, J. R. (1963). Factors influencing the metabolism of drugs in liver microsomes. Ann. N.Y. Acad. Sci. 104, 875-880. GAINES, T. B., AND KIMBROUGH, R. (1970). Oral toxicity of mirex in adult and suckling rats. Arch. Environ. Health 21, l-14. HALLETI-, D. J., NORSTROM, R. J., ONUSKA, F. I., COMBA, M. E., AND SAMPSON, R. (1976). Mass spectral confirmation and analysis by the Hall detector of mirex and photomirex in herring gulls from Lake Ontario. .I. Agr. Food Chem. 24, 1189-l 193. HALLETT, D. J., KHERA, K. S., STOLTZ, D. R., CHU, I., VILLENEUVE, D. C., AND TRIVETT, G. (1978). Photomirex: Synthesis and assessment of acute toxicity, tissue distribution, and mutagenicity. J. Agr. Food Chem. 26, 388-391. HUBER, J. J. (1965). Some physiological effects of the insecticide kepone in the laboratory mouse. Toxicol. Appt. Pharmacol. 7, 516-524. KHERA, K. S., VILLENEUVE, D. C., TERRY, G., PANOPIO, L., NASH, L., AND TRIVETT, G. (1976). Mirex: A teratogenicity, dominant lethal and tissue distribution study in rats. Food Cosmet. Toxicol. 14, 2529. SUNDARUM, A., VILLENEUVE, D. C., CHU, I., SECOURS,V. BECKING, G. C.. AND VALLI, V. E. (1980). Subchronic toxicity of photomirex in the female rat: Results of 28- and 90-day feeding studies. Drug Chem. Toxicol. 3, 105- 134. VASSEF, A. A. (1978). Direct micromethcd for colorimetry of some ornithine cambamoyl transferase activity with use of a linear standard curve. Clin. Chem. 24, 101-107. VILLENEUVE, D. C., RITTER, L., FELSKY, G., NORSTROM, R. J., MARINO, 1. A., VALLI, V. E., CHU, I., AND BECKING, G. C. (1979a). Short-term toxicity of photomirex in the rat. Toxicol. Appi. Pharmacol. 47, 105-I 14. VILLENEUVE, D. C., VALLI, V. E., CHU, I., SECOURS, V., RITTER, L., AND BECKING, G. C. (1979b). Ninety-day toxicity of photomirex in the male rat. Toxicology 12, 235-250. VILLENEUVE, D. C., KHERA, K. S., TRIVETT, G.. FELSKY, G., NORSTROM, R. J., AND CHU, 1. (1980). Photomirex: A tetratogenicity and tissue distribution study in the rabbit. J. Environ. Health Sci. 2, 171180. WARE, G. W., AND Goon, E. E. (1967). Effects of insecticides on reproduction in the laboratory mouse. II. Mirex, Tel&in, and DDT. Toxicol. Appl. Pharmacol. 10, 54-61.